DESCRIPTION: (Applicant's abstract) Endogenous nitric oxide (NO) mediates diverse functions in neuronal transmission and plasticity. In addition to its actions as a physiological messenger molecule, NO also participates in some forms of neurotoxic injury, including stroke and certain neurodegenerative processes. Nitric oxide synthase (NOS) is a calmodulin dependent enzyme and as such is regulated by the steep gradients of calcium encountered in the vicinity of open calcium channels. In brain NOS is functionally coupled to calcium influx through N-methyl-D-aspartate (NMDA) type glutamate receptors, while other calcium pools are poorly linked to NOS. A fundamental understanding of NO actions in the brain requires identification of the functional connection of nNOS with NMDA receptors. In an effort to address this important question, the applicant's laboratory has initiated a program of cell biological studies of NOS in brain. These experiments demonstrate that NOS is enriched at synaptic membranes owing to association of NOS with the postsynaptic density protein, PSD-95. Others have independently that PSD-95 clusters NMDA receptors at central synapses. The studies proposed in this grant seek to elucidate how these interactions with PSD-95 yield efficient coupling of calcium influx through NMDA receptors to NOS activity. Mutagenesis studies of the binding interfaces of NOS, PSD-95 and NMDA receptors will identify critical residues that confer binding specificity. Specific protein complexes containing NOS, PSD-95 and NMDA receptors will be identified in brain. These complexes will be reconstituted and functionally analyzed in transfected cells. The applicant will use various molecular techniques to disrupt PSD-95 in intact neurons. This will allow the applicant to determine the role of the PSD-95 complexes in coupling NMDA receptors to both NOS activity and neurotoxicity in vivo. Our studies will shed light on the molecular mechanisms that restrict NOS activity to specific calcium pathways in neurons. This work will be relevant to both mechanisms of synaptic plasticity and excitotoxic brain injury mediated by the NMDA receptor.